(a) Field of the Invention
The invention relates to a pharmaceutical agent(s) to induce cell death for use in treating conditions which involve inappropriate cell survival.
(b) Description of Prior Art
Replication of human adenoviruses in terminally differentiated epithelial cells requires an efficient mechanism to induce cellular DNA synthesis. This induction permits replication of viral DNA and production of progeny virus. Human adenoviruses infect and kill epithelial cells very efficiently. Cell death occurs by apoptosis and virus spread occurs through endocytosis by surrounding cells.
Products of early region 1A (E1A) of the adenovirus genome induce cell DNA synthesis and are largely responsible for cell transformation by adenoviruses. E1A produces two major mRNAs of 13S and 12S which encode proteins of 289 and 243 residues (289R and 243R, respectively) that are identical except for the lack in 243R of a central 46-amino acid sequence, termed conserved region 3 or CR3, as schematically depicted in FIG. 1A. Two additional regions present in the common sequence encoded by exon 1 of both E1A mRNAs are also conserved in all human adenovirus serotypes and have been termed CR1 and CR2. E1A products induce DNA synthesis through ,complex formation between CR2 and CR1 and the retinoblastoma tumor suppressor pRB and related p107 and p130 proteins, or between the amino terminus and CR1 and the transcriptional modulator p300 and possibly related proteins (Corbeil, H. B. et al., 1994, J. Virol. 68: 6697-6709). E1A-289R also activates expression of the early viral transcription units E2, E3, and E4, and certain cellular genes at least in part through interactions with transcription factors and basal transcription machinery requiring CR3 (Teodoro, J. G. et al., 1995, Oncogene 11: 467-474). In addition to CR3, transactivation of the E4 promoter has also been shown to rely to some degree on two regions encoded by the second exon of 13S mRNA, termed auxiliary regions 1 and 2, or AR1 and AR2. Production of stably transformed cells requires early region 1B (E1B) which encodes polypeptides of 19 and 55 kDa that are individually capable of cooperating with E1A via separate but additive pathways (McLorie, W. et al., 1991, J. Gen. Virol. 72: 1467-1471).
Considerable evidence indicates that a major function of E1B proteins in lytic infection and cell transformation is to suppress cytotoxic effects and apoptosis induced by expression of E1A. Without E1B, the toxicity of E1A products results in the death of E1A-transformed cells and a reduction in the yield of progeny due to the early demise of productively infected cells. E1A proteins can cause apoptosis by a process mediated by the tumor suppressor p53, which controls growth arrest and programmed cell death pathways (Teodoro, J. G. et al., 1995, Oncogene 11: 467-474). Expression of E1A products results in the elevation of p53 levels. The 55 kDa E1B protein binds to p53 and blocks both p53-mediated activation of gene expression and apoptosis (Teodoro, J. G. et al., 1994, J. Virol. 68: 776-786). The 19 kDa E1B protein appears to suppress apoptosis by a mechanism that is functionally analogous to that of the cellular proto-oncogene product Bcl-2 (Nguyen, M. et al., 1994, J. Biol. Chem. 269: 16521-16524). Cells infected with adenovirus mutants which fail to express the 19 kDa protein display enhanced cytotoxicity and extensive degradation of both cellular and viral DNA into nucleosome sized fragments (McLorie, W. et al., supra; Teodoro, J. G. et al., 1995, Oncogene 11: 467-474). At later times, even in the presence of E1B proteins, infected cells suffer apoptotic death and viral progeny spread to neighboring cells through endocytosis of cell fragments. In addition to the induction of DNA synthesis and cell transformation, the large 289-residue (289R) E1A protein also transactivates expression of all early viral genes, including early regions 1A, 1B, 2, 3 and 4 (reviewed in Teodoro, J. G. et al., 1995, Oncogene 11: 467-474).
It would be highly desirable to be provided with a pharmaceutical agent for induction of apoptosis when such induction is useful in the treatment of human diseases which involve inappropriate cell survival.
In accordance with the present invention, we have used a genetic approach to identify the role of individual E4 proteins in the induction of p53-independent apoptosis. Our results indicate the E4 death proteins, E4orf4 or E4orf6, are responsible for induction of p53-apoptosis in transformed, but not untransformed, cells. Thus, E4orf4 and E4orf6 are both powerful inducers of p53-independent cell death. This discovery has significant ramifications for both apoptosis-inducing therapeutics and drug screens.
In a first aspect, the invention provides a method of increasing apoptosis in a cell by administering to the cell an apoptosis inducing amount of an E4orf6 polypeptide or an apoptotic fragment thereof. In a preferred embodiment of this aspect, the apoptosis is p53-independent.
In a second aspect, the invention provides a method of increasing apoptosis in a mammal which includes providing a transgene encoding an E4orf6 polypeptide or an apoptotic fragment thereof to a cell of the mammal. The transgene is positioned for expression in the cell, and preferably encodes E4orf6.
In a third aspect, the invention provides a method of increasing apoptosis in a cell which includes administering to the cell a compound which increases E4orf6 biological activity. In various preferred embodiments, the compound is E4orf6 mRNA, or increases the stability of E4orf6.
In a preferred embodiment of the first and third aspects of the invention, the cell is in a mammal, preferably a human.
In a fourth aspect, the invention provides a method of increasing apoptosis in a cell by administering to the cell an apoptosis inducing amount of an E4orf4 polypeptide or an apoptotic fragment thereof. In a preferred embodiment of this aspect, the apoptosis is p53-independent.
In a fifth aspect, the invention provides a method of increasing apoptosis in a mammal which includes providing a transgene encoding an E4orf4 polypeptide or an apoptotic fragment thereof to a cell of the mammal. The transgene is positioned for expression in the cell, and preferably encodes E4orf4.
In a sixth aspect, the invention provides a method of increasing apoptosis in a cell which includes administering to the cell a compound which increases E4orf4 biological activity. In various preferred embodiments, the compound is E4orf4 mRNA, or increases the stability of E4orf4.
In a preferred embodiment of the fourth and sixth aspects of the invention, the cell is in a mammal, preferably a human.
In a seventh aspect, the invention provides a method of increasing apoptosis in a cell by administering to the cell an apoptosis inducing amount of a composition which includes an E4orf6 polypeptide or an apoptotic fragment thereof and an E4orf4 polypeptide or an apoptotic fragment thereof. In a preferred embodiment of this aspect, the apoptosis is p53-independent.
In an eighth aspect, the invention provides a method of increasing apoptosis in a mammal which includes providing a first transgene encoding an E4orf6 polypeptide or fragment thereof and a second transgene encoding an E4orf4 polypeptide or fragment thereof to a cell of the mammal. The first and second transgenes are positioned for expression in the cell and, preferably, encode E4orf6 and E4orf6, respectively.
In a ninth aspect, the invention provides a method of increasing apoptosis in a cell which includes administering to the cell a composition which includes a first compound which increases E4orf6 biological activity and a second compound which increases E4orf4 biological activity. In various preferred embodiments, the first compound is E4orf6 mRNA, or increases stability of E4orf6, and the second compound is E4orf4 mRNA or increases stability of E4orf4.
In a preferred embodiment of the seventh and ninth aspects of the invention, the cell is in a mammal, preferably a human.
In preferred embodiments of all the above aspects of the invention, the cell is in a mammal diagnosed as having a disease involving insufficient apoptosis. Preferably, the disease is cancer.
In a tenth aspect, the invention features a pharmaceutical composition which includes substantially pure nucleic acid encoding an E4orf6 polypeptide and a pharmaceutically acceptable carrier. In one embodiment of this aspect, the nucleic acid encodes E4orf6 having a conservative amino acid substitution relative to the E4orf6 sequence of FIG. 15 (SEQ ID NO.: 2).
In an eleventh aspect, the invention features a pharmaceutical composition which includes nucleic acid encoding an apoptotic fragment of E4orf6.
In a preferred embodiment of the tenth and eleventh aspects of the invention, the nucleic acid is in a viral vector. In another embodiment, the nucleic acid is operably linked to regulatory sequences for expression of the polypeptide and the regulatory sequences include a promoter. In another embodiment, the promoter is a constitutive promoter, is inducible by one or more external agents, or is cell-type specific.
In a twelfth aspect, the invention features a pharmaceutical composition which includes a nucleic acid having the sequence of FIG. 15 (SEQ ID NO.: 1), or degenerate variants thereof, and encoding the amino acid sequence of FIG. 15 (SEQ ID NO.: 2).
In a thirteenth aspect, the invention features a pharmaceutical composition which includes nucleic acid having about 50% or greater nucleotide sequence identity to the DNA sequence of FIG. 15 (SEQ ID NO.: 1), where the nucleic acid encodes a polypeptide with E4orf6 apoptotic biological activity. In one embodiment of this aspect of the invention, the nucleotide sequence identity is 75% or greater to the DNA sequence of FIG. 15 (SEQ ID NO.: 1).
In a fourteen aspect, the invention features a pharmaceutical composition which includes a DNA sequence substantially identical to the DNA sequence of FIG. 15 (SEQ ID NO.: 1).
In a fifteenth aspect, the invention features pharmaceutical composition which includes substantially pure mammalian E4orf6 polypeptide, or apoptotic fragment thereof. In one embodiment of this aspect, the polypopetide includes an amino acid sequence substantially identical to the amino acid sequence shown in FIG. 15 (SEQ ID NO.: 2). In another embodiment, the polypeptide has a conservative amino acid substitution relative to the E4orf6 sequence of FIG. 15 (SEQ ID NO.: 2).
In a sixteenth aspect, the invention features a pharmaceutical composition which includes a substantially pure polypeptide fragment of E4orf6.
In a seventeenth aspect, the invention features a pharmaceutical composition which includes substantially pure nucleic acid encoding an E4orf4 polypeptide and a pharmaceutically acceptable carrier. In one embodiment of this aspect, the nucleic acid encodes E4orf4 having a conservative amino acid substitution relative to the E4orf4 sequence of FIG. 16 (SEQ ID NO.: 4).
In an eighteenth aspect, the invention features a pharmaceutical composition which includes nucleic acid encoding an apoptotic fragment of E4orf4.
In a preferred embodiment of the seventeenth and eighteenth aspects of the invention, the nucleic acid is in a viral vector. In another embodiment, the nucleic acid is operably linked to regulatory sequences for expression of the polypeptide and the regulatory sequences include a promoter. In another embodiment, the promoter is a constitutive promoter, is inducible by one or more external agents, or is cell-type specific.
In a nineteenth aspect, the invention features a pharmaceutical composition which includes a nucleic acid having the sequence of FIG. 16 (SEQ ID NO.: 3), or degenerate variants thereof, and encoding the amino acid sequence of FIG. 16 (SEQ ID NO.: 4).
In a twentieth aspect, the invention features a pharmaceutical composition which includes nucleic acid having about 50% or greater nucleotide sequence identity to the DNA sequence of FIG. 16 (SEQ ID NO.: 3), where the nucleic acid encodes a polypeptide with E4orf4 apoptotic biological activity. In one embodiment of this aspect of the invention, the nucleotide sequence identity is 75% or greater to the DNA sequence of FIG. 16 (SEQ ID NO.: 3).
In a twenty-first aspect, the invention features a pharmaceutical composition which includes a DNA sequence substantially identical to the DNA sequence of FIG. 16 (SEQ ID NO.: 3).
In a twenty-second aspect, the invention features pharmaceutical composition which includes substantially pure mammalian E4orf4 polypeptide, or apoptotic fragment thereof. In one embodiment of this aspect, the polypopetide includes an amino acid sequence substantially identical to the amino acid sequence shown in FIG. 16 (SEQ ID NO.: 4). In another embodiment, the polypeptide has a conservative amino acid substitution relative to the E4orf4 sequence of FIG. 16 (SEQ ID NO.: 4).
In a twenty-third aspect, the invention features a pharmaceutical composition which includes a substantially pure polypeptide fragment of E4orf4.
In the twenty-fourth and twenty-fifth aspects, the invention features methods for identifying a compound as an E4orf6 analog or an E4orf4 analog which includes first providing a cell expressing the adenovirus E1A-289R protein while not expressing any E4 proteins. The cell is then contacted with a candidate compound and cell viability is determined, where death in the cell indicates a compound that is an E4orf6 or an E4orf4 analog.
In one embodiment of the twenty-fourth and twenty-fifth aspects of the invention, the cell is selected from the group consisting of: 1A.A3, 1A.A6, and 1A.A12 cells. In other preferred embodiments, the viability is measured with Trypan Blues(trademark), a DNA fragmentation assay, an Annexin V binding assay, or Propidium Iodide, or a combination thereof. In yet another embodiment, the cell is infected with a mutant adenovirus incapable of expressing any E4 proteins.
In a twenty-sixth aspect, the invention features a method for identifying a compound as an E4orf4 analog which includes first providing a cell expressing protein phosphatase 2A. The cell is then contacted with the compound the activity of the protein phosphatase 2A in the cell is measured, where an increase in the activity relative to a cell not contacted indicates a compound that is an E4orf4 analog.
In a twenty-seventh aspect, the invention features a pharmaceutical agent for induction of apoptosis for the treatment of human diseases which involve inappropriate cell survival, which includes E4orf6, an analog, or a biologically active fragment thereof.
In a twenty-eighth aspect, the invention features a pharmaceutical composition for the treatment of human diseases which involve inappropriate cell survival, which includes a therapeutical amount of E4orf6, an analog, or a biologically active fragment thereof in association with a pharmaceutical carrier.
In a twenty-ninth aspect, the invention features a pharmaceutical composition for the treatment of human diseases which involve inappropriate cell survival, which includes a therapeutic amount of a compound which induces apoptosis or other cytotoxic effects analogous to E4orf6 biological activity in association with a pharmaceutical carrier.
In a thirtieth aspect, the invention features a pharmaceutical agent for induction of apoptosis for the treatment of human diseases which involve inappropriate cell survival, which includes E4orf4, an analog, or a biologically active fragment thereof.
In a thirty-first aspect, the invention features a pharmaceutical composition for the treatment of human diseases which involve inappropriate cell survival, which includes a therapeutic amount of E4orf4, an analog, or a biologically active fragment thereof in association with a pharmaceutical carrier.
In a thirty-second aspect, the invention features a pharmaceutical composition for the treatment of human diseases which involve inappropriate cell survival, which includes a therapeutic amount of a compound which induces protein phosphates 2a in association with a pharmaceutical carrier. In one embodiment of this aspect, the compound is an agonist of E4orf4. In another embodiment, the compound mimics E4orf4 activity.
In a thirty-third aspect, the invention features a pharmaceutical composition for the treatment of human diseases which involve inappropriate cell survival, which includes a therapeutic amount of a compound which induces apoptosis or other cytotoxic effects analogous to E4orf4 biological activity in association with a pharmaceutical carrier.
In a thirty-fourth aspect, the invention features a pharmaceutical agent for induction of apoptosis for the treatment of human diseases which involve inappropriate cell survival, which includes E4orf6, an analog, or a biologically active fragment thereof; and E4orf4, an analog, or a biologically active fragment thereof.
In a thirty-fifth aspect, the invention features a pharmaceutical composition for the treatment of human diseases which involve inappropriate cell survival, which includes a therapeutical amount of E4orf6, an analog, or a biologically active fragment thereof; and E4orf4, an analog, or a biologically active fragment thereof in association with a pharmaceutical carrier.
In a thirty-sixth aspect, the invention features a pharmaceutical composition for the treatment of human diseases which involve inappropriate cell survival, which includes a therapeutic amount of a compound which induces apoptosis or other cytotoxic effects analogous to biological activities of the E4 death proteins in association with a pharmaceutical carrier.
Compositions of the invention include, but are not limited to, E4orf4 protein, E4orf6 protein, combinations thereof, nucleic acids encoding the E4orf4 and E4orf6 polypeptides, analogs, mimetics, and any agonist therapeutic agents identified using any of the methods disclosed herein. The compositions may be administered with a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer such compositions to patients.
In accordance with the present invention, the expression xe2x80x9cdiseases which involve inappropriate cell survivalxe2x80x9d includes, without limitation, diseases caused by HIV, herpes and/or other viral infections, Alzheimer""s disease, cancer, arthritis, and lupus.
In accordance with the present invention, xe2x80x9cE4 death proteinsxe2x80x9d include products encoded by DNA capable of hybridizing at high stringency conditions to nucleic acids encoding E4orf6 (SEQ ID NO.: 1) and E4orf4 (SEQ ID NO.: 3), provided in FIGS. 15 and 16 respectively, and which also have E4orf6 and/or E4orf4 biological activity. Products are encoded by DNA that is at least 500 nucleotides in length, preferably, less than 200 nucleotides in length, more preferably, less than 150 nucleotides in length, and most preferably, less than 100 nucleotides in length. It will be understood that E4orf6 and E4orf4 proteins and nucleic acids of the invention may be obtained from any adenovirus strain having the E4orf6 or E4orf4 open reading frames, as defined as an open reading frame which is at least 20%, preferably 50%, more preferably 75%, and most preferably 90% identical to the E4orf6 (SEQ ID NO. 2) and E4orf4 (SEQ ID NO.: 4) open reading frames, respectively, provided herein.
In accordance with the present invention, xe2x80x9cE4orf6 proteinsxe2x80x9d and xe2x80x9cE4orf6 polypeptidesxe2x80x9d include products encoded by DNA capable of hybridizing at high stringency conditions to nucleic acids encoding E4orf6 (SEQ ID NO.: 1) provided in FIG. 15, and which also have E4orf6 biological activity. Products are encoded by DNA that is at least 500 nucleotides in length, preferably, less than 200 nucleotides in length, more preferably, less than 150 nucleotides in length, and most preferably, less than 100 nucleotides in length. It will be understood that E4orf6 proteins and nucleic acids of the invention may be obtained from any adenovirus strain having the E4orf6 open reading frame, as defined as an open reading frame which is at least 20%, preferably 50%, more preferably 75%, and most preferably 90% identical to the E4orf6 open reading frame (SEQ ID NO.: 2) provided herein.
In accordance with the present invention, xe2x80x9cE4orf4 proteinsxe2x80x9d and xe2x80x9cE4orf4 polypeptidesxe2x80x9d include products encoded by DNA capable of hybridizing at high stringency conditions to nucleic acids encoding E4orf4 (SEQ ID NO.: 3), provided in FIG. 16, and which also have E4orf4 biological activity. Products are encoded by DNA that is at least 500 nucleotides in length, preferably, less than 200 nucleotides in length, more preferably, less than 150 nucleotides in length, and most preferably, less than 100 nucleotides in length. It will be understood that E4orf4 proteins and nucleic acids of the invention may be obtained from any adenovirus strain having the E4orf4 open reading frame, as defined as an open reading frame which is at least 20%, preferably 50%, more preferably 75%, and most preferably 90% identical to the E4orf4 open reading frame (SEQ ID NO.: 4) provided herein.
In accordance with the present invention, the expression xe2x80x9chigh stringency conditionsxe2x80x9d means conditions that allow DNA hybridization to nucleic acids encoding E4orf6 (SEQ ID NO.: 1) or E4orf4 (SEQ ID NO.: 3) at high stringency (e.g., hybridizing in 2xc3x97SSC at 40xc2x0 C. with a DNA probe length of at least 40 nucleotides). For other definitions of high stringency conditions, see Ausubel, F. et al., 1994, Current Protocols in Molecular Biology, John Wiley and Sons, New York, 6.3.1-6.3.6, hereby incorporated by reference.
In accordance with the present invention, the expression xe2x80x9cE4orf6 biological activityxe2x80x9d means the ability to induce in E1A-289R expressing cells an increased cell death that is 25%, more preferably 40%, and most preferably 60% greater than the cell death observed in E1A-289R expressing cells not expressing E4orf6, an apoptotic fragment, or analog thereof. E4orf6 biological activity is determined using one of the assays provided herein, preferably the luciferase death assay using 1A.A3, 1A.A6, or 1A.A12 cells. It will be understood that E1A-289R may be from any adenovirus strain.
In accordance with the present invention, the expression xe2x80x9cE4orf4 biological activityxe2x80x9d means the ability to induce in E1A-289R expressing cells an increased cell death that is 50%, more preferably 75%, and most preferably 90% greater than the cell death observed in E1A-289R expressing cells not expressing E4orf4, an apoptotic fragment, or analog thereof. E4orf6 biological activity is determined using one of the assays provided herein, preferably the luciferase death assay using 1A.A3, 1A.A6, or 1A.A12 cells. It will be understood that E1A-289R may be from any adenovirus strain.
In accordance with the present invention, the expression xe2x80x9cpromoterxe2x80x9d means a minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell type-specific, tissue-specific or inducible by external signals or agents; such elements may be located in the 5xe2x80x2 or 3xe2x80x2 regions of the native gene.
In accordance with the present invention, the expression xe2x80x9cdegenerate variantxe2x80x9d means nucleic acid sequences or combinations thereof selected from all possible coding sequences for E4orf6 and E4orf4, or polypeptide fragments thereof, based upon the universal genetic code.
In accordance with the present invention, the expression xe2x80x9coperably linkedxe2x80x9d means that a gene and one or more regulatory sequences are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.
In accordance with the present invention, the expression xe2x80x9cpositioned for expressionxe2x80x9d means that the nucleic acid is positioned adjacent to a nucleic acid sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, e.g., an E4orf4 polypeptide, a recombinant protein or a RNA molecule).
In accordance with the present invention, the expression xe2x80x9csubstantially identicalxe2x80x9d means a polypeptide or nucleic acid exhibiting at least 50%, preferably 75%, more preferably 90%, and most preferably 95% identity to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably at least 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 70 nucleotides, more preferably at least 90 nucleotides, and most preferably at least 120 nucleotides.
Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). This software program matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
By xe2x80x9ctransformed cellxe2x80x9d is meant a cell which is immortalized. For example, a transformed cell will divide and give rise to two daughter cells of the same differentiation status as the parent cell. A transformed cell may be a cell into which (or into an ancestor of which) has been introduced an exogenous gene or gene product (e.g., an oncogene) which allows immortalization of that cell. A transformed cell may also arise from a genomic mutation in an endogenous gene, giving rise to a mutated gene product, or dysregulation of a endogenous gene product. Transformed cells are differentiated from stem cells in that transformed cells have an alteration affecting normal gene expression and/or regulation. Exemplary transformed cells include cancerous cells such as those found in solid and liquid tumors.
In accordance with the present invention, the expression xe2x80x9ctransgenexe2x80x9d means any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
In accordance with the present invention, the expression xe2x80x9ctransgenicxe2x80x9d means any cell which includes a nucleic acid sequence which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell. As used herein, the transgenic cells are generally transgenic mammalian cells and the nucleic acid (transgene) is inserted by artifice into the nuclear genome.
In accordance with the present invention, the expression xe2x80x9cpolypeptidexe2x80x9d means any chain of more than two amino acids, regardless of post-translational modification, such as glycosylation or phosphorylation. Polypeptides include proteins, polypeptide fragments thereof, peptide mimetics thereof, and mutants thereof.
In accordance with the present invention, the expression xe2x80x9capoptotic fragmentxe2x80x9d means a polypeptide fragment of an E4 death protein (i.e., E4orf4 and E4orf6) that has a transformed cell-killing ability that is 75%, more preferably 95%, or most preferably, 100% or greater when compared to the transformed cell-killing ability of the full length protein.
In accordance with the present invention, the expression xe2x80x9csubstantially pure polypeptidexe2x80x9d means a polypeptide that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the polypeptide is an E4 death protein polypeptide that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure. A substantially pure E4 death protein polypeptide may be obtained, for example, by extraction from a natural source (e.g. an adenovirus) by expression of a recombinant nucleic acid encoding an E4 death protein polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
A protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include those derived from adenoviruses but synthesized in E. coli or other prokaryotes. By xe2x80x9csubstantially pure nucleic acidxe2x80x9d is meant nucleic acid (e.g., DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a DNA or a DNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
In accordance with the present invention, the expression xe2x80x9cspecifically bindsxe2x80x9d is meant an antibody that recognizes and binds a protein but that does not substantially recognize and bind other molecules in a sample, e.g., a biological sample, that naturally includes protein. Preferably, the specifically binding antibody which specifically binds an adenovirus protein (e.g., E4orf4) does not bind another adenovirus protein (e.g., E4orf6).
In accordance with the present invention, the expression xe2x80x9canalogxe2x80x9d includes, without limitation, polypeptide fragments, peptide and non-peptide mimetics, reagents and compounds which mimic the cell killing function, and reagents and compounds which mimic other functions of E4orf4 or E4orf6 proteins.
In accordance with the present invention, the expression xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d means a carrier which is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations arc known to one skilled in the art and described, for example, in Remington""s Pharmaceutical Sciences, (18th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa.
In accordance with the present invention, the expression xe2x80x9cviral vectorxe2x80x9d means a strand of DNA which includes elements taken from a virus. Viral vectors may direct the expression of inserted DNA from an exogenoous promoter, or may direct expression of inserted DNA from the vector""s own long terminal repeat (LTR) sequences. Preferable viral vectors are able to be packaged in non-lytic viruses capable of infecting cells which then expressed the DNA inserted into the viral vector.
The pharmaceutical agent of the present invention allows for the selective killing of cells that are prevented from dying by a virus or as a consequence of a disease state. Thus, the pharmaceutical agent of the present invention only kills the inappropriately surviving cells, such as cancer cells or viral infected cells. This results in a substantially side effect free therapy for the patient.
The pharmaceutical agent of the present invention includes, without limitation, E4 death proteins of any adenovirus of any serotype, fragment thereof, and peptide and non-peptide mimetics of these protein products.